This invention relates to methods and reagents for marking strains of microorganisms and cells and for the identification of genes.
The following is a general discussion of the relevant art, none of which is admitted to be prior art to the invention.
Bacterial infections of host organisms create difficulties in a variety of different fields, notably in human medicine. In order to develop effective treatments to control such bacterial infections, it is frequently important to understand the mechanisms involved in the pathogenesis process. Therefore, it is useful to identify and isolate the genes involved in pathogenesis, which can also be used as targets in various methods for the identification and development of anti-bacterial drugs.
Several different approaches and methods have been used to identify bacterial genes involved in pathogenesis. The various approaches seek to identify pathogenesis-related genes, based on one or more characteristics linking the expression of the gene with the pathogenesis process. Thus, various approaches seek to identify sets of genes, such as genes encoding various toxins and protein factors involved in binding to and invading host cells, genes that are preferentially expressed in vivo (e.g., by differential display, differential hybridization, or by use of "In vivo Expression Technology", IVET), and genes that are required for in vivo survival and growth. While the methods previously used for these approaches have been able to identify some pathogenesis related genes, those methods have limitations as described below:
1. By isolating genes encoding toxins and other known virulence factors, the regulation of these genes and their roles in the pathogenesis process can be studied in more detail. Identification of genes encoding exotoxins and other readily-recognized genes requires substantial effort in investigation of the gene products and in establishing their role in pathogenesis. In addition, many genes involved in pathogenesis are not exotoxins, nor are they readily recognized as virulence factors. Thus, many genes which are specifically expressed in vivo and/or are essential for in vivo survival or growth cannot be identified by this approach. PA1 2. Differential display examines mRNAs that are specifically present after in vivo growth or after growth under conditions that mimic the in vivo environment. This method requires that a particular in vivo specific mRNA be present at a relatively high level to be detected, which may not always occur. In addition, the presence of large amounts of rRNA and other RNAs can often reduce the power of this technique. PA1 3. The IVET technology likewise identifies genes which are preferentially expressed in vivo, and has been used to identify many such genes (Mahan et al., 1993, Science 259:686-688). However, most of the genes isolated by this method are merely housekeeping genes and thus are not useful as targets for anti-bacterial therapy. Furthermore, since IVET identifies the in vivo expressed genes by the ability of their promoter to direct expression of a selectable gene involved in specific nutrient synthesis or antibiotic resistance, the promoters must be strong enough to be identified. Consequently, in vivo expressed genes with weak promoters will fail to be identified in this method. Finally, IVET technology does not provide mutants useful in establishing a direct role in pathogenesis for the in vivo expressed gene. PA1 4. To isolate genes that are essential for in vivo survival/growth, a method of using transposons to tag and mutagenize cells was developed (Hensel et al., 1995, Science 269:400-403). In this method, a mixed population of such mutagenized cells is grown and the mutants that fail to survive and grow in vivo are detected by the disappearance of the corresponding specific oligonucleotide tag. The corresponding gene is then identified as it is the transposon-interrupted gene for that mutant strain. While new in vivo essential genes have been identified in Salmonella typhimurium using this method, several factors limit its use in a range of bacteria under different conditions. PA1 a. Not all mutagenesis methods can be efficiently applied to every type organism. For example, random and efficient transposon mutagenesis systems have not been observed or developed in many bacteria. It is difficult to apply site directed mutagenesis in bacterial strains where the genetics and molecular biology has not been developed. Different chemical and physical (e.g., UV) mutagens may have different killing and mutagenizing effects on different organisms depending on their cell wall and cell membrane structures, DNA compositions, DNA repair systems, etc. Certain mutagens and/or mutagenizing methods may be more suitable than others for a given organism. Therefore, having available a large array of mutagenesis methods to choose from broadens the application of this invention in various organisms. PA1 b. Different kinds of mutations can be generated by using different mutagenesis methods. These include point mutations (such as missense and nonsense mutations and those in the regulatory regions), insertions, and deletions. The mutagenesis methods can be targeted to certain gene(s) or even to certain nucleotides, such as in vitro site-directed mutagenesis, mutagenesis by error-prone PCR and DNA chemical synthesis, and knockout mutants generated by integration and other homologous recombination events. Other mutagenesis methods are rather random, targeting the whole genome, such as many transposons and most chemical and physical mutagens. It has been known that even for mutagens that induce random mutations, their modes of action are quite different from each other, thus generating different types of mutations. Mutations in certain gene(s) having detectable phenotypes may be obtained by one mutagen but not by others. The more mutagenesis methods available, the more likely that a desired mutant form(s) of a gene can be generated. Therefore, it will be especially advantageous if one has the ability to choose different mutagenesis means to mutagenize and identify a large number of genes whose mutant forms share a common phenotype. For example, in searching for genes essential for in vivo growth by transposon mutagenesis, if one such gene is upstream of an in vitro essential gene in the same operon, a transposon insertion in the in vivo gene will greatly diminish or completely block the expression of its downstream in vitro essential gene. This will make it difficult to obtain mutants in the in vivo gene by transposon insertion because mutants are not able to be propagated in vitro due to the polar effect. On the other hand, it is possible to obtain point mutations, such as missense mutations, in the in vivo essential gene without the polar effect, by other means of mutagenesis, e.g., chemical mutagens or UV irradiation.
First, as transposons are used as the tool for self-tagging and mutagenesis, the method cannot be used in bacteria which do not possess a random insertion transposon system. This prevents the use of this method in many medically important bacteria or in other pathogens such as fungi and viruses.
Second, even in organisms with developed random transposon technology, the only type of mutants generated by this method are transposon-insertional mutants. This excludes, or at least severely limits, the use of other mutagens to generate other types of mutants.
Third, the use of relatively large amounts of radioactive material in producing labeled probes, and the laborious procedures of DNA hybridization and detection make this method difficult, slow, expensive, and environmentally unfavorable.
Fourth, the presence, in some organisms, of "hot spots" for transposon insertion (a relatively common phenomenon) and cross-reactivity among oligonucleotide tags can reduce the effectiveness of the screen and create interpretive difficulties.